Decreased ACKR3 (CXCR7) function causes oculomotor synkinesis in mice and humans

Abstract

Oculomotor synkinesis is the involuntary movement of the eyes or eyelids with a voluntary attempt at a different movement. The chemokine receptor CXCR4 and its ligand CXCL12 regulate oculomotor nerve development; mice with loss of either molecule have oculomotor synkinesis. In a consanguineous family with congenital ptosis and elevation of the ptotic eyelid with ipsilateral abduction, we identified a co-segregating homozygous missense variant (c.772G>A) in ACKR3, which encodes an atypical chemokine receptor that binds CXCL12 and functions as a scavenger receptor, regulating levels of CXCL12 available for CXCR4 signaling. The mutant protein (p.V258M) is expressed and traffics to the cell surface but has a lower binding affinity for CXCL12. Mice with loss of Ackr3 have variable phenotypes that include misrouting of the oculomotor and abducens nerves. All embryos show oculomotor nerve misrouting, ranging from complete misprojection in the midbrain, to aberrant peripheral branching, to a thin nerve, which aberrantly innervates the lateral rectus (as seen in Duane syndrome). The abducens nerve phenotype ranges from complete absence, to aberrant projections within the orbit, to a normal trajectory. Loss of ACKR3 in the midbrain leads to downregulation of CXCR4 protein, consistent with reports that excess CXCL12 causes ligand-induced degradation of CXCR4. Correspondingly, excess CXCL12 applied to ex vivo oculomotor slices causes axon misrouting, similar to inhibition of CXCR4. Thus, ACKR3, through its regulation of CXCL12 levels, is an important regulator of axon guidance in the oculomotor system; complete loss causes oculomotor synkinesis in mice, while reduced function causes oculomotor synkinesis in humans.

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Figures

Figure 1

Family with oculomotor synkinesis segregates a homozygous ACKR3 c.772G>A (p.Val258Met) variant. (A–D) Photographs of affected participant (IV-9) showing bilateral ptosis in primary gaze (A) with lid elevation with ipsilateral abduction (B,C) and minimal lid elevation on upgaze (D). (A–D) reprinted with permission from Khan et al., JAAPOS 2004. (E) Family pedigree. Black-filled circles indicate affected individuals: IV-3 has bilateral congenital ptosis, IV-9 has bilateral congenital ptosis with lid elevation on abduction, IV-13 has left sided ptosis with ipsilateral lid elevation on abduction and IV-14 has right-sided ptosis with ipsilateral lid elevation on abduction. All are homozygous for the ACKR3 c.772G>A (p.Val258Met) variant. The half-sister filled in gray (IV-1) has isolated Duane syndrome without ptosis. She is heterozygous for the variant. The parents (III-2 and III-3) are heterozygous for the variant and are unaffected by any oculomotor abnormality. Genotypes for the ACKR3 variant and chromosome 2 polymorphic markers are shown underneath each individual. Two-point linkage analysis at D2S338 reveals a LOD score of 3.3. (F) Sanger sequencing of ACKR3 shows c.772G in control, heterozygous c. 722G>A in the unaffected mother and homozygous c.722G>A in an affected individual. (G) Valine258 is conserved in mammals and X. tropicalis, but not D. rerio, in which the gene is duplicated. (H) Schematic of ACKR3. Val258 is in the sixth transmembrane domain (red). Extracellular cysteines are labeled in yellow. K208, D179 and D275 (purple) are necessary for CXCL12 binding (33) (see Discussion). Residues labeled in pink were mutated in (33) but did not decrease CXCL12 binding. Residues labeled in green have homozygous missense variants present in the gnomad database.

Figure 2

V258M ACKR3 is expressed and traffics to the cell surface, but it has a lower Kd for CXCL12 binding. HEK293 cells were transiently transfected with plasmids encoding wild-type human ACKR3 (A), V258M human ACKR3 (B) or empty vector (C), fixed after 24 h and stained with anti-human ACKR3 antibody (red). Expression and localization of the mutant protein is indistinguishable from wild-type. Binding affinity for CXCL12 was tested using an HTRF assay (see Materials and Methods for explanation). Kd for the wild-type receptor is 0.2028 nM ±0.06792 (mean ± SEM), similar to published reports of the Kd for ACKR3 and CXCL12 binding (7, 27, 28). Kd for the mutant receptor is 3.814 ± 1.214 (P = 0.0267, paired t-test, n = 6).

Figure 3

Expression of Ackr3 mRNA in region of developing mouse oculomotor nucleus. In situ hybridization of Isl1 on wild-type (A, C) and Ackr3KO/KO (E) tissue shows Isl1 expression in developing oculomotor neurons at E11.5 (A, E) and E13.5 (C). In situ hybridization of Ackr3 on wild-type (B, D) and Ackr3KO/KO (F) tissue shows Ackr3 expression in wild-type Isl1-expressing CN3 neurons and surrounding tissues at E11.5 (B) and E13.5 (F) and absence of expression in Ackr3KO/KO tissue (H). All Isl1 and Ackr3 pairs are adjacent sections. Scale bar in (G) equals 100 µm for all sections.

Figure 4

Ackr3 knock-out mice display variable misrouting of CN3, CN4 and CN5. (A) E11.5 maximum intensity projections of whole mount imaging of an IslMN:GFP wild-type embryo, counterstained with anti-smooth muscle actin to label muscles and arteries (red), demonstrates the normal trajectory of the oculomotor (CN3), trochlear (CN4), abducens (CN6), motor trigeminal (CN5m) and facial (CN7) nerves. Midbrain to orbit of one side of embryo enlarged in (B). (D) E11.5 Ackr3KO/KO embryo. Oculomotor axons project dorsally in the midbrain and stall. CN7, CN9, CN10 and CN12 have grossly normal trajectories, while spinal cord axons project dorsally and aberrantly. There is variability in CN3, CN4, CN5m and CN6 trajectories, as displayed in (D), (E) and (G–I). In (D) and (E), all oculomotor axons project dorsally and stall, while in (G) and (H), axons from the caudal portion of the oculomotor nucleus project dorsally, but the rostral portion forms a thin CN3, which projects to the orbit. The arrow in (H) shows an abnormal branch of CN3. In 8/24 orbits, axons of CN5m aberrantly project toward the orbit [arrow in (G)]. CN6 reaches the orbit in 8/24 orbits (E), stalls in 4 (G) and cannot be identified in 12/24 orbits (D, H). (C) Photomicrograph of a whole E11.5 wild-type embryo. (F) Two embryos displayed severe malformations of the back of the head. In these embryos, CN4 is severely misrouted, reaching the midline, but not crossing and instead projecting caudally (I). Images in (B), (E) and (G–I) have been cropped in the z dimension to show only one side of the head. N = 12 Ackr3KO/KO and 10 wild-type controls. Scale bars: 500 µm (A, D, I) and 200 µm (B, E, G, H).

Figure 5

Loss of Ackr3 leads to aberrant midline crossing of CN3 axons rostrally. (A) Dorsal view of CN3 and CN4 in an E11.5 wild-type IslMN-GFP embryo, counterstained with anti-smooth muscle actin to label muscles and arteries (red), reconstructed and cropped using Imaris software from a whole mount embryo imaged sagitally. CN3 projects ventrally (into the page, with no midline crossing). CN4 projects dorsolaterally and crosses the midline before projecting contralaterally. (B) Dorsal view of an E11.5 Ackr3KO/KO embryo shows rostral midline crossing of CN3 axons (arrow). CN4 projects normally. Scale bar: 400 µm.

Figure 6

Aberrant innervation patterns of CN3 and CN6. (A) View of orbit in an E13.5 IslMN:GFP:Hb9:GFP embryo, counterstained with anti-smooth muscle actin to label muscles and arteries (red), reconstructed and cropped using Imaris software from a whole mount embryo imaged sagitally, shows the normal innervation patterns of CN3, CN4 and CN6. CN3 enters the orbit, the superior division branches to the SR (yellow arrowhead), an inferior decision region forms between the MR and IR (yellow arrow), and a branch extends to the IO. CN4 projects to the SO. CN6 projects to the LR. (B) Example of an Ackr3KO/KO with Duane syndrome pathology. CN6 is absent. CN3 is thin, has scant projections to the SR and aberrant branches to the LR (yellow arrow). (C) Central orbital view of an E13.5 Hb9:GFP wild-type embryo shows the normal trajectory of CN6 to the LR. (D) Example of an E13.5 Ackr3KO/KO embryo in which CN6 not only innervates the LR muscle but also has an aberrant branch that divides to innervate other EOMs that are normally innervated solely by CN3 (yellow arrow). Scale bar in (A) equals 200 µm in (A, B). Scale bar in (C) equals 200 µm in (C, D). SR, superior rectus; SO, superior oblique; MR, medial rectus; IR, inferior rectus; IO, inferior oblique; LR, lateral rectus.

Figure 7

The facial nerve branches normally, but cell bodies in the facial nucleus fail to migrate. (A) Sagittal view of hindbrain in an E13.5 IslMN:GFP embryo reconstructed and cropped using Imaris software from a whole mount embryo imaged sagitally shows the trajectory of the facial nerve, inner ear efferents and the migrating cell bodies (yellow arrow). (B) In an E13.5 Ackr3KO/KO embryo, the facial nerve and IEEs have normal projections from the hindbrain, but the cell bodies do not migrate (yellow arrow). (C) Sagittal view of an E14.5 IslMN:GFP embryo shows the normal branching pattern of CN7. (D) In an E13.5 Ackr3KO/KO embryo, the facial nerve shows normal peripheral branching. Scale bar in (A) equals 200 µm for (A, B) and scale bar in (C) equals 300 µm for (C, D).

Figure 8

CXCR4 protein, but not mRNA, is downregulated with loss of Ackr3. In situ hybridization for Isl1 (A) and Cxcr4 (B) and immunohistochemistry for IslMN:GFP (C, E; green) and CXCR4 (D, E; red) at E11.5 shows Cxcr4 expression in and around CN3. In Ackr3KO/KO embryos, in situ hybridization for Isl1 (F) and Cxcr4 (G), and RT-PCR for Cxcr4 (K) shows normal levels of Cxcr4 mRNA, but immunohistochemistry for IslMN:GFP (H, J; green) and CXCR4 (I, J; red) and western blot of whole brain for CXCR4 (L, M) show very low levels of CXCR4 protein expression. (A) and (B) are adjacent sections, as are (F) and (G). Scale bar in (A) equals 100 µm for (A, B, F, G). Scale bar in (H) equals 100 µm for (C–E, H–J). In (K), differences are not significant (P = 0.69). In (M), asterisks indicate P < 0.001.

Figure 9

Excess CXCL12 causes CN3 axon misrouting, similar to inhibition of CXCR4. Time-lapse imaging of slice cultures from E10.5 IslMN:GFP embryos at 0, 12, 24 and 36 h in culture. In wild-type embryos, (top) over the course of 36 h CN3 (green) grows toward the eyes and branches extensively. When 50 or 100 nM CXCL12 is added (middle panels), axons exit the oculomotor nucleus dorsally. Those that had already exited continue toward the eye. This phenotype is strikingly similar to the phenotype when 1 µg/ml AMD3100 (specific inhibitor for CXCR4, bottom) is added. Bottom panel reprinted with permission from Whitman et al. IOVS 2018. D, dorsal; V, ventral; E, eye; N, oculomotor nucleus; arrow, oculomotor axons (white: wild-type projection, yellow: aberrant projection). Scale bar equals 200 µm.